1. Field of the Invention
The present invention relates generally to a method for site-directed mutagenesis and more particularly to a method for site-directed mutagenesis of more than, for example, 10 sites simultaneously with up to 100% efficiency.
2. Description of the Prior Art
Site-directed mutagenesis is a powerful tool to explore the structure-function relationship of proteins, but most traditional methods rely on the mutation of only one site at a time and efficiencies drop drastically when more than three sites are targeted simultaneously. Many applications in functional proteomics and genetic engineering, including codon optimization for heterologous expression, generation of cysteine-less proteins or alanine-scanning mutagenesis, would greatly benefit from a multiple-site mutagenesis method with high efficiency.
Probing the structure-function relationship of proteins and nucleic acids by site-directed mutagenesis has become an important strategy in functional studies and genetic engineering, see J. Braman (Ed.), In Vitro Mutagenesis Protocols, Methods in Molecular Biology, Vol. 182, 2nd Ed, Humana Press, Totowa, 2002, the entire contents and disclosure of which is hereby incorporated by reference. In site-directed mutagenesis, the genetic code for a protein is altered at a specific site by changing one (or multiple) of the individual nucleotides that make up a gene and that code for a specific protein. A variety of very efficient methods have become available for site-directed mutagenesis of individual sites, both PCR-based and non-PCR-based, and several convenient commercial kits are on the market, see T. A. Kunkel, Rapid and efficient site-specific mutagenesis without phenotype selection, Proc. Natl. Acad. Sci. USA, 82 (1985) pp. 488-492; M. P. Weiner, G. L. Costa, W. Schoettlin, J. Cline, E. Mathur, J. C. Bauer, Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction, Gene, 151 (1994) pp. 119-123; and T. M. Ishii, P. Zerr, X. M. Xia, C. T. Bond, J. Maylie, J. P. Adelman, Site-directed mutagenesis, Methods Enzymol., 293 (1998) pp. 53-71, the entire contents and disclosures of which are hereby incorporated by reference. Most of these methods rely on the mutation of only one site at a time and efficiencies drop drastically when more than three sites are targeted simultaneously. For example, Sawano and Miyawaki provided a protocol that allows for the introduction of mutations at two sites simultaneously with one primer for each mutation site, which required two rounds of PCR amplification separated by an additional DpnI digest of parental DNA. Nevertheless, they achieved an efficiency of 76% for only two mutations, but efficiencies drop further at an exponential rate if more than three sites are targeted in a single step, see A. Sawano and A. Miyawaki, Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis, Nucleic Acids Research, Vol. 28, No. 16 (2000) p. e78, the entire contents and disclosure of which is hereby incorporated by reference.
Several multiple site-directed mutagenesis methods have also been described, which all require multiple rounds of PCR, including successive rounds of overlap extension PCR or successive rounds of PCR combined with in vitro dam-methylation/ligation before and DpnI digest and gel purification after each PCR step, see I. Michaelian, A. Sergeant, A general and fast method to generate multiple site directed mutations, Nucleic Acids Res., 20 (1992) p. 376; U. N. Dwivedi, N. Shiraishi, W. H. Campbell, Generation of multiple mutations in the same sequence via the polymerase chain reaction using a single selection primer, Anal. Biochem., 221 (1994) pp. 425-428; K. S. Bhat, Multiple site-directed mutagenesis, Methods Mol. Biol., 57 (1996) pp. 269-277; A. R. Meetei, M. R. Rao, Generation of multiple site-specific mutations in a single polymerase chain reaction product, Anal. Biochem., 264 (1998) pp. 288-291; Y. G. Kim, S. Maas, Multiple site mutagenesis with high targeting efficiency in one cloning step, Biotechniques, 28 (2000) pp. 196-198; G. C. Lee, L. C. Lee, V. Sava, J. F. Shaw, Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris, Biochem. J., 366 (2002) pp. 603-611; and international application numbers WO 03/002761A1 and WO 99/25871, the entire contents and disclosures of which are hereby incorporated by reference. Nevertheless, these methods are complicated by the time requirements for multiple PCR rounds, combined with an increased risk of introducing undesired second-site mutations during extensive use of thermostable polymerases in repeated PCR rounds. Furthermore, most of these methods require two complementary mutagenic primers for each site-directed mutation. Hence, a rapid and efficient multiple-site mutagenesis method would be of great benefit to a variety of applications in functional proteomics and genetic engineering that, for example, require codon optimization for heterologous expression systems, the generation of cysteine-less proteins for subsequent cysteine-scanning mutagenesis and disulfide-scanning mutagenesis studies, or (re)design and removal of restriction endonuclease sites in expression vectors, genes of selectable markers, etc., see J. A. Javitch, L. Shi, G. Liapakis, Use of the substituted cysteine accessibility method to study the structure and function of G protein-coupled receptors, Methods Enzymol., 343 (2002) pp. 137-156; and M. A. Danielson, R. B. Bass, J. J. Falke, Cysteine and disulfide scanning reveals a regulatory alpha-helix in the cytoplasmic domain of the aspartate receptor, J. Biol. Chem., 272 (1997) pp. 32878-32888, the entire contents and disclosures of which are hereby incorporated by reference.